|
| Status |
Public on Jul 04, 2006 |
| Title |
viremic HIV patient D |
| Sample type |
RNA |
| |
|
| Source name |
Monocytes from viremic (OFF HAART) HIV patient D
|
| Organism |
Homo sapiens |
| Characteristics |
Monocytes isolated from an aviremic HIV infected patient; aviremia due to successful suppression of HIV by HAART.
|
| Treatment protocol |
PBMCs were isolated using Ficoll and stored in Liquid N2. Cells were thawed and peripheral blood CD14+ cells were isolated by fluorescence-activated cell sorting, using a FACSVantage SE FACSDiVa flow cytometer (BD Immunocytometry Systems, San Jose, CA). Frozen peripheral blood mononuclear cells were thawed and lymphocytes were stained according to the manufacturer’s instructions with the monoclonal antibodies CD3 FITC (BD Immunocytometry Systems San Jose, CA), CD4 PC5 (Beckman Coulter, Miami, FL), and CD14 PE (Becton Dickenson). Sorting for CD14+ monocytes was performed using a quadrant gate on CD3 FITC-CD14 PE+. CD14+ monocyte purity was determined by flow cytometry to be >95%.
|
| Growth protocol |
Cells were not cultured i.e. total RNA isolated following cell sorting.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen RNAEasy extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
One half to two micrograms of total RNA was isolated from 1-2 million CD14+ sorted cells. Target RNA was labeled (as biotinylated cRNA) as described in the Affymetrix 2003 Technical Note “GeneChip Eukaryotic Small Sample Target Labeling Assay Version II”.
|
| |
|
| Hybridization protocol |
Following fragmentation, 10 microg of cRNA were hybridized for 16 hr at 45C on the Human U133A GeneChip. The GeneChips was washed and stained using the Affymetrix Fluidics Station 400 following Manufacturer's recommended protocol..
|
| Scan protocol |
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
|
| Description |
Monocytes isolated from an aviremic HIV infected patient; aviremia due to successful suppression of HIV by HAART.
|
| Data processing |
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 150.
|
| |
|
| Submission date |
Jul 03, 2006 |
| Last update date |
Jul 03, 2006 |
| Contact name |
Richard A Lempicki |
| E-mail(s) |
rlempicki@mail.nih.gov
|
| Phone |
301-846-5093
|
| Organization name |
Leidos Biomedical Research, Inc.
|
| Department |
Clinical Services Program
|
| Lab |
Laboratory of Immunopathogenesis and Bioinformatics
|
| Street address |
PO Box B
|
| City |
Frederick |
| State/province |
MD |
| ZIP/Postal code |
21702 |
| Country |
USA |
| |
|
| Platform ID |
GPL96 |
| Series (1) |
| GSE5220 |
Longitudinal comparison of monocytes from an HIV viremic vs avirmeic state |
|
